PROJECT SUMMARY/ABSTRACT Systemic administration of high-dose IL-2 has been used since the 1980?s as an FDA-approved immunotherapy for metastatic cancer. Despite the fact that up to 9% of patients treated with high dose IL-2 achieve a durable, long term response, this therapy is rarely used today due to significant life-threatening complications. Such complications occur due to IL-2 activation of vascular endothelium, resulting in systemic capillary leak, as well as other adverse effects associated with ?off target? signaling of IL-2. In addition, IL-2 preferentially activates CD4+Foxp3+ regulatory T cells that mitigate the tumor-specific response. We recently published an IL-2 fusion protein which targets and solely activates cytotoxic T lymphocytes in the absence of endothelial or regulatory T cell activation. In addition to a dramatically increased safety profile, our IL-2 fusion protein inhibits the growth of highly aggressive tumors normally resistant to other forms of immunotherapy. The purpose of the current proposal is to evaluate the immunogenicity of this protein, explore its clinical response when used in combination with checkpoint blockade immunotherapy, and evaluate its utility for expansion of tumor-reactive leukocytes for adoptive cell therapy. Successful completion of the proposed studies will support a Phase II STTR application and eventually advancement of this therapy to IND-enabling studies. Our ultimate goal is to submit this novel immunotherapy to the FDA for first-in-human clinical safety trials.